Novel methods and kits for detecting of urea cycle disorders using mass spectrometry

Abstract

The present invention relates to newborn screening kits, methods, stable isotopically-labeled internal standards or internal standard solution for high throughput screening and analysis of metabolic disorders using liquid chromatography mass spectrometry (LC-MS) are provided. The metabolic disorders can be amino acid, organic acid or fatty acid oxidation disorders, and particularly urea cycle disorders or deficiencies, hyperammonemia, Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria. The newborn screening kits, methods, stable isotopically-labeled internal standards or internal standard solution are particularly useful for newborn screening (NBS) of metabolic disorders.

What is claimed is: 
     
         1 . A method of detecting the presence and/or measuring the levels of one or more metabolites in a sample obtained from a subject, by tandem mass spectrometry, said metabolites comprising at least glutamine and said method comprising the following steps:
 (i) extracting said metabolites from the sample with an extraction solution;   (ii) providing, before or after the step (i), a solution comprising a known amount of one or more a stable isotopically-labeled internal standard corresponding to said metabolites to be detected in the sample,   (iii) ionizing said metabolites and said one or more stable isotopically-labeled internal standards to generate ions,   (iv) acquiring the mass to charge (m/z) ratio of said one or more ions in the mass spectrum in the Multiple Reaction Monitoring (MRM) mode,   (v) determining the sum of glutamine and lysine in MRM mode, and   (vi) detecting and quantifying the amount of glutamine with stable isotopically-labeled internal standard corresponding to lysine.   
     
     
         2 . The method according to  claim 1  for diagnosis of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria, further comprising a step (vii) wherein elevated level of glutamine as obtained in step (vi) is detected compared to physiological level of glutamine. 
     
     
         3 . The method according to  claim 1 , wherein one or more metabolites are selected among amino acids, organic acids, acylcarnitines, a plurality of carnitines, and/or succinylacetone are further detected and quantified in said sample, and wherein said method further comprise a step (v) for determining the presence and/or the amounts of said one or more metabolites by analyzing the mass MRM spectrum as obtained in step (iv). 
     
     
         4 . The method according to  claim 3 , wherein said amino acids comprise aspartate, serine, proline, phenyalanine, tyrosine, methionine, valine, leucine, citrulline, homocitrulline, ornithine, arginine, alanine, N-acetyl glutamate, glycine, argininosuccinate and/or oxoproline, and/or wherein said organic acid comprises orotic acid, or said acyl carnitines or plurality of carnitines comprise free carnitines, acetyl carnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarninitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and/or stearoylcarnitine. 
     
     
         5 . The method according to  claim 1 , wherein the stable isotopically-labeled internal standard solution comprises at least a lysine stable isotopically-labeled internal standard, and further comprises one or more stable isotopically-labeled internal standards corresponding to alanine, arginine, citrulline, glycine, leucine, methionine, ornithine, phenylalanine, glutamine valine, lysine, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetyl carnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarninitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and/or stearoylcarnitine, and wherein said one or more stable isotopically-labeled internal standards are dried. 
     
     
         6 . The method according to  claim 1 , wherein said sample is a body fluid sample, preferably a blood sample, and more preferably a dried blood sample, and/or wherein said subject is a subject suspected of having a metabolic disorder, such as a neonate, a newborn, or a child, preferably a newborn. 
     
     
         7 . The method according to  claim 1 , wherein said ionizing step (iii) is performed by delivering said extracted metabolites and standards to an ion source of the mass spectrometer by liquid chromatography system, and/or wherein said data acquisition step (iv) is performed with a triple-quadrupole mass spectrometer or high-resolution mass spectrometer, and/or wherein the step (v) is performed by selecting at least one accurate mass-to-charge (m/z) ratio ions corresponding to an at least one calculated mass-to-charge (m/z) ratio of said one or more metabolites present in said sample. 
     
     
         8 . The method according to  claim 2 , wherein the determined amount or presence of glutamine correlates with the presence or absence of urea cycle disorder, and/or hyperammoneamia, and/or Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria. 
     
     
         9 . A newborn screening kit for detecting the presence and/or measuring the levels of glutamine in a blood sample of a newborn, by tandem mass spectrometry, according to the method of  claim 1 , comprising an internal standard solution comprising at least a stable isotopically-labeled internal standard corresponding to lysine. 
     
     
         10 . Newborn screening kit according to  claim 9 , for the diagnosis of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria. 
     
     
         11 . Newborn screening kit according to  claim 9 , wherein said internal standard solution further comprises one or more stable isotopically-labeled internal standards corresponding to one or more metabolites selected among amino acids, organic acids, acylcarnitines, a plurality of carnitines, and/or succinylacetone. 
     
     
         12 . Newborn screening kit according to  claim 9 , wherein said internal standard solution further comprises one or more stable isotopically-labeled internal standards corresponding to alanine, arginine, citrulline, homocitrulline, glycine, leucine, methionine, ornithine, phenylalanine, valine, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, glutamic acid, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetyl carnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarninitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and/or stearoylcarnitine, wherein said standards are optionally dried, and wherein said kit allows for the diagnosis in said newborns or of one or more metabolic deficiencies selected among urea cycle disorders, and/or hyperammonemia, Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH), and/or argininosuccinic aciduria, as well as further metabolic newborn disorders, selected among amino acid disorders, fatty acid oxidation (FAO) disorders, and organic acidurias (OA). 
     
     
         13 . Newborn screening kit according to  claim 9 , wherein said sample is a body fluid sample, preferably a blood sample, and more preferably a dried blood sample, and/or wherein said newborn is suspected of having a metabolic disorder. 
     
     
         14 . Newborn screening kit according to  claim 9 , further comprising at least one plate or microplate, dried blood spot controls, extraction solution, eluent solution, and/or one or more covers. 
     
     
         15 . Newborn screening kit according to  claim 9 , which comprises one or more of the following elements:
 one or more extraction plates;   one or more analysis plates;   dried blood spot controls;   stored stable isotopically-labeled internal standards of known concentrations (labeled amino acids);   extraction solution;   eluent solution;   adhesive plastic covers; and/or   optionally one or more aluminium foil covers.   
     
     
         16 . Newborn screening kit according to  claim 9 , wherein said stored stable isotopically-labeled internal standards of known concentrations comprise labeled amino acids, and/or labeled acylcarnitines, and/or labeled succinyl acetone; and/or labeled argininosuccinic acid, wherein said internal standards are stored in separate vials or in a common vial, and wherein said internal standards are optionally dried. 
     
     
         17 . Newborn screening kit according to  claim 9 , further comprising a technical sheet or instructions or protocol steps for the screening, wherein said steps comprise of preparation of a working solution comprising said one or more reconstituted labeled internal standards with the extraction solution, and a step of sample extraction, wherein said steps may be performed in any order. 
     
     
         18 . Newborn screening kit according to  claim 9 , further comprising instructions for the following steps:
 a step of preparation of internal standards (labeled amino acids, labeled acylcarnitines, etc.),   a step 2 of preparation of a working solution comprising reconstituted labeled internal standards with the extraction solution, and/or   a step of sample extraction, and   
       optionally further comprising instruction for instrument settings according to the instrument manufacturer, and/or for calculation of the results according to the instrument manufacturer. 
     
     
         19 . An internal standard solution as defined in the method of  claim 1 , comprising at least a lysine stable isotopically-labeled internal standard, and further comprising one or more stable isotopically-labeled internal standards corresponding to alanine, citrulline, homocitrulline glycine, leucine, methionine, ornithine, glutamine, phenylalanine, valine, glutamic acid, aspartic acid, glutamic acid, tyrosine, serine, oxoproline, proline, argininosuccinate, succinylacetone, orotic acid, free carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, isovalerylcarninitine, glutarylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, and/or stearoylcarnitine, wherein said standards are optionally dried. 
     
     
         20 . Internal standard solution of  claim 19 , wherein said lysine stable isotopically-labeled internal standard is  13 C 6 - 15 N 2 -lysine, and said further stable isotopically-labeled internal standards are  2 H 4 -alanine,  2 H 2 -citrulline,  15 N- 13 C-glycine,  2 H 3 -leucine, 2H 3 -methionine, 2H 6 -ornithine,  13 C 6 -phenylalanine,  2 H 8 -valine,  2 H 3 -aspartic acid,  2 H 3 -glutamic acid,  13 C 3 -serine,  13 C 6 -tyrosine,  2 H 5 -proline,  13 C 5 -succinylacetone,  15 N 4 - 13 C 6 -argininosuccinate,  2 H 9 -carnitine,  2 H 3 -acetylcarnitine,  2 H 3 -propionylcarnitine,  2 H 3 -butyrylcarnitine,  2 H 9 -isovalerylcarnitine,  2 H 3 -glutarylcarnitine,  2 H 3 -hexanoylcarnitine,  2 H 3 -octanoylcarnitine,  2 H 3 -decanoylcarnitine,  2 H 3 -lauroylcarnitine,  2 H 9 -myristoylcarnitine,  2 H 3 -palmitoylcarnitine, and/or  2 H 3 -stearoylcarnitine, wherein said standards are optionally dried.